Studies on HIV pathogenesis and preclinical evaluation of HIV-1 gene therapeutic constructs would be greatly facilitated by the use of a suitable animal model that can mimic the physiological conditions afforded by a normally functioning human hematopoietic system. As the previously used xenograft SCID-hu mouse models lacked the capacity for generating primary immune responses, viral pathogenic effects on the human cells could only be evaluated in the context of the absence of an active immune response. Thus an important physiological dimension was missing in these systems. This deficiency can now be potentially rectified by the use of a newly emerged immunocompetent humanized mouse model. Injection of human CD34 hematopoietic stem cells into immunodeficient neonatal Rag2-/-yc-/- mice results in multi-lineage human hematopoiesis and the generation of a functional immune system. Recent evidence showed that these humanized mice are permissive for disseminated HIV infection. With the capacity for multi-lineage human cell engraftment and susceptibility for HIV infection, this model shows great potential to evaluate anti-HIV gene therapeutic constructs in a stem cell-based setting. Gene modified cells can now be tested for their capacity for immune reconstitution. We recently made progress in the construction and evaluation of several promising anti-HIV RNA inhibitory molecules such as siRNAs and propose to utilize this new mouse model to answer several important questions relevant for the success of gene therapy approaches. Our specific aims are to: 1. Further develop the humanized Rag2 common gamma chain double knock out (Rag2-/-yc-/-) mouse model for HIV infection to evaluate anti-HIV gene therapeutic constructs, 2. Evaluate the engraftment potential and multi-lineage differentiation of anti-HIV gene transduced CD34 hematopoietic progenitor cells in humanized Rag2-/-yc-/- mice, 3. Determine the expression of anti-HIV genes in terminally differentiated T cells and macrophages and evaluate their resistance to HIV-1 challenge in vitro and in vivo. 4. Evaluate the functional competence of transgenic hematopoietic cell subsets and the capacity of humanized Rag2-/-yc-/- mice bearing transgenic cells to give rise to normal immune responses: These studies will be helpful in developing new animal models for HIV/AIDS research, and in evaluating novel gene therapy approaches to control HIV infection.